Studies from the last several years have repeatedly highlighted the importance of long noncoding RNAs (lncRNA) in epigenetic regulation, development, and disease. However, the mechanisms by which RNAs control these processes remain poorly defined. Improved understanding of RNA-based mechanisms is crucial, especially given that 70-80% of the mammalian genome is transcribed and that the vast bulk of transcription is noncoding. Nowhere is the abundance of lncRNAs more evident than at the X-inactivation center (Xic), an X-linked region that controls the X-chromosome inactivation (XCI) in the female mammal. XCI serves as an excellent model to study lncRNA regulation because this process is controlled by a series of RNA-based switches. Silencing is initiated by the 17-kb Xist RNA as it recruits Polycomb proteins to the X-chromosome. Xist RNA is in turn controlled by an antisense transcript, Tsix, which antagonizes Xist by repelling the recruitment of an RNA-protein complex containing Polycomb proteins. My laboratory has also found that Xist upregulation requires the action of two additional lncRNAs, RepA which recruits Polycomb proteins to the Xic, and Jpx which is required to activate Xist transcription. The GM58839 grant has, for the past 16 years, provided crucial support for this lncRNA research. Herein, we propose to advance our understanding of RNA regulation by studying how RNAs of the Xic control the different steps and forms of XCI. Specifically, we will: (i) Investigate how lncRNAs play a role in homologous X-chromosome pairing, a process proposed to regulate counting and choosing of X-chromosomes for inactivation; (ii) Determine the mechanism by which Jpx RNA activates Xist expression; and (iii) Elucidate fundamental differences between random and imprinted XCI. The proposed work will require five years to complete and is expected to train three postdoctoral fellows for academic careers in the fields of epigenomics, RNA regulation, and stem cell biology.